ANTICANCER RESEARCH 35: 2531-2538 (2015)
Fast, Stable Induction of P-Glycoprotein-mediated Drug Resistance in BT-474 Breast Cancer Cells by Stable Transfection of ABCB1 Gene YUAN TANG1, YUE WANG2, SUDHIR DEOSARKAR1, FARIBORZ SOROUSH1, MOHAMMAD F. KIANI1 and BIN WANG1,3 1Department
of Mechanical Engineering, Temple University, Philadelphia, PA, U.S.A.; and Medical Oncology, Lankenau Medical Center, Wynnewood, PA, U.S.A.; 3Department of Biomedical Engineering, Widener University, Chester, PA, U.S.A.
2Hematology
Abstract. Background: Patients with P-glycoprotein and HER2/neu (HER2) receptor-overexpressing breast cancer usually have poor clinical outcomes. However, there exist no commercially available breast cancer cell lines that are HER2/P-glycoprotein double-positive, which limits research in this field. Materials and Methods: We report on the development and characterization of a drug-resistant sub-line from an HER2-positive breast cancer cell line by stable transfection of the ATP-binding cassette (ABC) subfamily B member 1 (ABCB1) gene which encodes P-glycoprotein. Results: ABCB1 gene expression levels were higher after transfection, which led to a 40-fold increase in P-glycoprotein expression. Interestingly, the transfection of ABCB1 also led to a slight increase in HER2 gene and protein expression levels. The transfection of ABCB1 increased the Pglycoprotein expression levels significantly. Conclusion: The method used herein for developing this cell line is appropriate for fast, stable induction of P-glycoprotein-mediated drug resistance compared to traditional methods. The in vitro cytotoxicity test suggests this cell line has cross-resistance to a wide range of chemotherapeutic agents. Breast cancer is the most common cancer in women (1). Research has shown that ~25-30% of breast tumors overexpress HER2/neu (HER2) receptor (1-3). These breast tumors tend to be much more aggressive and fast-growing (4), therefore, patients with elevated HER2 levels usually have poor clinical outcomes compared to patients with HER2-negative disease (5).
Correspondence to: Bin Wang, One University Place, Chester, PA 19013, U.S.A. Tel: +1 6104994089, Fax: +1 6104994059, e-mail:
[email protected] Key Words: MDR, HER2, P-glycoprotein, transfection, breast cancer, BT-474.
0250-7005/2015 $2.00+.40
Chemotherapy is a major therapeutic approach for the treatment of localized and metastasized breast cancer. The major problem in the management of HER2-overexpressing breast cancer is its multidrug resistance (MDR), especially in patients with recurrent breast cancer (6). HER2 overexpression has been found to correlate with tumor growth and metastasis, as well as with the development of chemoresistance (7, 8). As evident from both laboratory and clinical studies, HER2 may render tumor cells resistant to a wide range of chemotherapeutic agents, such as paclitaxel, cyclophosphamide, methotrexate, 5-fluorouracil, and epirubicin (9-11). ATP-binding cassette (ABC) subfamily B member 1 (ABCB1) gene-mediated overexpression of P-glycoprotein (P-gp) has been recognized as one of the major mechanisms in drug resistance in breast cancer (12). The 170-kDa ABCB1 gene encodes transmembrane P-gp (14, 15). Although the exact mechanism is still not fully understood, ABCB1 gene appears to play a key role in the development of drug resistance in breast cancer both in vitro and in vivo (13-18). P-gp resides in the cell membrane and can actively transport chemotherapeutic agents out of cancer cells using energy from ATP hydrolysis, leading to accelerated drug efflux and decreased net drug accumulation in tumor (19). Although the role of HER2 in breast cancer has been extensively studied over the years, some of the underlying mechanisms of MDR development in the HER2-positive subtype are still far from being completely understood (20). Research in this area has been hampered, in part, by the fact that currently there exist no commercially available breast cancer cell lines that are both HER2- and P-gp-positive, which limits the scope of research in this field. We report on the development and characterization of a drug-resistant subline from a HER2-positive breast cancer cell line by stable transfection of the ABCB1 gene. This new drugresistant cell line has the potential to serve as a model cell
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ANTICANCER RESEARCH 35: 2531-2538 (2015) line for studying targeted-drug delivery of chemotherapeutic agents using antibody-tagged immunoliposomes.
Materials and Methods Cell culture and transfection. HER2 overexpressing (2) human breast carcinoma cell line BT-474 (ATCC, Manassas, VA, USA) was cultured as monolayers in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (Invitrogen, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/ streptomycin (Invitrogen) in a humidified atmosphere of 95% air and 5% CO2 at 37˚C. Cells were subcultured twice weekly. For experiments, the cells were grown in plastic tissue culture flasks (FisherSci, Pittsburg, PA, USA) and used when in the exponential growth phase. Unlike traditional transfection methods involving virus vector (21), BT-474 cells were transfected with ABCB1 gene packaged in a plasmid vector (EX-E2266-M02; Genecopoeia, Rockville, MD, USA) using Lipofectamine 2000 tranfection reagent (Invitrogen) according to the manufacturer’s instructions. After transfection, cells were maintained in culture media containing 60 ng/ml of colchicine to select P-gp-positive cells. Western blot. Cultured cells were washed three times with ice-cold phosphate buffered saline (PBS) and lysed in a lysis buffer cocktail containing protease inhibitor (50 mmol/L Tris-HCl (pH 7.5), 150 mmol/l NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mmol/l EDTA, 1 mmol/l sodium fluoride, 1 mmol/l sodium orthovanadate, 1 mmol/l phenylmethylsulfonyl fluoride, 10 μl/ml phosphatase inhibitor, and 10 μl/ml protease inhibitor cocktail (all purchased from SigmaAldrich, St. Louis, MO, USA) for 30 min on ice. Proteins (15 μg for HER2; 50 μg for ABCB1) were separated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) and transferred onto a nitrocellulose membrane (BioRad, Hercules, CA, USA). The membrane was blocked with 5% nonfat dry milk plus 0.1% Tween 20 in Tris-buffered saline (TBS) and then incubated with primary antibodies (mouse anti-human ABCB1 monoclonal antibody, SC-55510; rabbit anti-human HER2/neu polyclonal antibody, SC-284; both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4˚C. Membranes were washed three times with TBS containing 0.1% Tween20 and then incubated for 1 h with secondary antibodies in blocking buffer. The signal was detected with a SuperSignal West Pico Chemiluminescent kit (Thermo Scientific, Rockford, IL, USA). Real-time reverse transcription polymerase chain reaction. ABCB1 and HER2 gene expression level was quantified using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) technique. Cells were first lysed using Trizol reagent (Life Technologies) to extract RNA according to the manufacturer’s instructions. RNA concentrations were measured in a Nanodrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). First-strand cDNA was synthesized from total RNA using the GoTaq® 2-Step RT-qPCR System (Promega, Madison, WI, USA). Relative mRNA levels were than measured with a realplex EP gradient S mastercycler (Eppendorf, Hauppauge, NY, USA) through the SYBR Green detection system. The relative amount of expression of each gene was calculated as the ratio of the studied gene to the control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
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Figure 1. Relative mRNA levels of HER2/neu (HER2) and ATP-binding cassette (ABC) subfamily B member 1 (ABCB1) transfected into BT-474 cells compared to non-transfected BT-474 cells using 2(-ΔΔCt) method, normalized to a control gene glyceraldehyde 3-phosphate dehydrogenase. *p
